Hi guys, I am an undergrad being introduced to a lot of different lab techniques, so I'm not entirely familiar with what is going on. We recently ligated a gene into a pBS vector and popped it into some E. colicells. We looked for positive clones via blue/white screening and isolated/purified the respective plasmid. Then, we carried out a PCR reaction of the pBS-gene (our clone). This PCR reaction is something I do not really understand.

We used the pBS-gene as our template. We carried out four reactions, which are as followed:

Here is an image of the vector we used: http://www.bio.davidson.edu/courses/mol ... ipt%2B.gif. On the right hand side, it says, "MCS" (where our gene of interest is located), of which is "flanked" by the T3 and T7 promoters. The thing that throws me off is just where these primers will bind to, and what the subsequent product will be. The one that really throws me off is number 2, because it seems like we might not get a reaction? Are number 1 and 2 just positive and negative controls?

Based on the few PCR reactions I tried drawing out myself (sorry, don't have an image), from my understanding, the amplified products I should get are:

1) T3 - coding region - T72) ??? It looks like it WON'T be a proper PCR reaction? Will we just get one massive chunk of DNA? 3) coding region - T74) T3 - coding region

I think the point of this entire process was to just determine the directionality of our insert. Because later in the lab, we did a whole bunch of sequence alignments with our gene (of both strands) and BLASTed it. But...I am just not really sure if I've done the PCR reactions correctly.

2) if your gene is inserted into the vector so the beginning of the gene is right next to T7: T7-gene.If your gene is inserted into the vector so that the beginning of the gene is next to T3: Nothing since both primers bind in the same direction.

3) if your gene is inserted into the vector so the beginning of the gene is right next to T7: Nothing since both primers bind in the same direction.If your gene is inserted into the vector so that the beginning of the gene is next to T3: T7-gene.

4) if your gene is inserted into the vector so the beginning of the gene is right next to T7: Nothing since both primers bind in the same direction.If your gene is inserted into the vector so that the beginning of the gene is next to T3: gene-T3.